Rewriting E. coli’s Genetic Code

Researchers use directed evolution to create a bacterial strain that substitutes a synthetic base for thymine.

Aug 5, 2011

Sabine LouÃ«t

E. coli cells that use synthetic base 5-chlorouracile in place of thymineIG-CEA

Using a new automated system for placing strong selective pressure on cultured bacteria, a team of European scientists has evolved a new strain of E. coli that almost completely lacks thymine—one of the four bases of the DNA alphabet. Instead, the bacteria contain the structurally similar synthetic base, 5-chlorouracil.

The results, published in a recent issue of Angewandte Chemie International Edition, suggest a new way for incorporating unnatural elements into living organisms—a technical challenge in synthetic biology that has yet to see industrial applications.

“They have shown that they have a good control of the evolution process,” said molecular geneticist George Church of Harvard University, who was not involved in the research. “Darwin would be very proud!”

Organisms evolve as a result of randomly-occurring mutations that provide a fitness advantage (or disadvantage) over their peers, allowing them to send more (or fewer) offspring into the next generation. Scientists involved in synthetic biology have often attempted to speed up the pace of microbial evolution in a lab, as a way to engineer organisms to perform useful functions, for example. Most of these efforts applied a mutagen or insert mutated DNA, then selected for those bacteria that exhibit the traits of interest.

But these methods based on molecular biology add a layer of complexity that makes them less attractive for industrial applications. So Philippe Marlière, CEO of biotechnology company Heurisko USA Inc., and his colleagues decided to step back from targeted mutations and instead focus on the selection process to alter the chemical composition of E. coli.

Specifically, they used an automated system that monitored the population density of the bacteria at set intervals and supplied the culture with a variable supply of thymine and 5-chlorouracil—an unnatural base that is normally toxic for the organisms. When the density surpassed a given threshold, the cells received an injection of 5-chlorouracil. Should the population density fall below a minimum threshold, on the other hand, the bacteria would receive an injection of thymine. The result was a culture that was constantly exposed to sublethal levels of 5-chlorouracil. As the bacteria evolved tolerance to the substance, the system automatically upped the injections of 5-chlorouracil, but never so high as to kill the whole population. In essence, the system was selecting for genetic variants capable of tolerating higher and higher concentrations of the toxic substance.

“It is a bit like a trainer putting an athlete on a treadmill with a heart monitor,” explained Church. “As the athlete gets stronger, the trainer pushes the treadmill.”

Because 5-chlorouracil is so similar to thymine, with a single chlorine replacing thymine's methyl group, as the cells evolved tolerance to the synthetic base, they began to incorporate it into their DNA during replication. After about 1,000 generations, which took around 140 days, the researchers had evolved an E. coli strain in which 5-chlorouracil had replaced 98 percent of the thymine. “We over-estimated the resistance from nature,” said Marlière. “In 5 months, we managed to change the chemical composition of the bacteria.”

Importantly, the bacteria were healthy, growing at a sustainable rate. By comparison, previous attempts for such a base substation had only achieved about 90 percent replacement rate, and had hampered bacterial growth.

Furthermore, because the system is fully automated and operates with large populations of E. coli cells, it can be easily scaled up for future potential industrial applications. While the current experiment was “a scientific proof of concept,” Marlière said, that has not been implemented in industry yet, the researchers ultimately hope to generate new organisms that harbor metabolic traits optimized for bioremediation, alternative modes of energy production, or for the synthesis of high-value chemicals on an industrial scale.